In every cell the genome operates as a three-dimensional integrative unit where different chromosomes occupy distinct territories or compartments within a nucleus but where the precise architecture and functional consequences of such organization remain elusive. We hypothesize that physical interactions between distant chromatin regions do occur in a neuron-specific manner and contribute to establishment of unique neuronal phenotypes and plasticity within a circuit. As a result, the preexisting three-dimensional (3-D) position coding can be a factor in genome-wide integration of the activity of thousands of genes, including establishing crucial epigenetic marks, and can be one of the mechanisms coordinating the complex transcriptional output of a cell. The major aims of this proposal are (1) to map long-range interactions of the cellular genome in synaptically coupled identified neurons and, (2) to characterize the dynamics of the 3-D reorganization of the nuclear genome following standard learning tests and synaptic stimulation. Here, using large accessible sensory, modulatory and motor neurons of the simpler defensive circuit in Aplysia, we will implement a novel Hi-C (chromosome conformation capture) approach to probe the 3-D architecture of the whole genome at the level of single neurons. The method is based on the combination of proximity-based ligation and selective capture of distinct anatomical regions of a nucleus with massive parallel sequencing. Thus, we will map interactive regions of the neuronal genome both in control conditions and following well established long-term plasticity tests (such as 5-HT applications). First, such spatial mapping of the genome conformation will allow us to unbiasedly characterize the location within a single nucleus of distinct mutually interacting chromatin compartments. Second, we will correlate their positions (e.g. central vs. peripheral) to the expression level of genes and their regulatory regions located within these compartments. Finally, we will correlate the expression level of selected genes with 5-cytosine methylation patterns (methylome) within gene regulatory regions, focusing upon components of 5-HT mediated signal transduction. This approach can be extended to other epigenetic marks (e.g. using chromatin immunoprecipitation for selective histone posttranslational modification events as activation and repression marks respectively) to probe mechanisms of integrative activity of neurons following synaptic inputs or drug administration. This paradigm can serve as a powerful proof-of-concept platform to characterize mechanisms of this most elusive cellular and genomic process leading to integrative activity of neurons, with broad implications to fundamental and clinical studies from drug abuse mechanisms to memory research. PUBLIC HEALTH RELEVANCE: Knowing the spatial organization of DNA methylation and transcriptional units within functionally characterized neurons is crucial for understanding the mechanisms of integrative activity of neurons. Indeed, in every cell the genome operates as a three-dimensional integrative entity where different chromosomes occupy distinct territories or compartments within a nucleus. Yet physical interactions between distant chromatin regions do occur to regulate gene activity, form epigenetic marks and coordinate complex transcriptional output of a cell. Here, we will characterize the 3-D architecture of long-range interactions of the cellular genome and its dynamics in uniquely identified neurons as they learn and remember. Information about positional coding within the nuclear genome is central to developing targeted therapies for the broad spectrum of pathological processes associated with drug abuse and memory loss.